Epitope recognized by anti-interferon gamma autoantibodies in patients with disseminated mycobacterial infections and the application therefor

ABSTRACT

The present invention discloses a fragment of peptide which can be utilized in patients suffering from a disseminated mycobacterial infection. The fragment of peptide contains a sequence of amino acids with seven residues as formula (I) shown below, wherein X1 is Leucine (Leu); X2 is Proline (Pro); X3 is Glutamate (Glu); X4 is serine (Ser); X5 is Serine (Ser); X6 is Leucine (Leu) and X7 is Arginine (Arg): X1-X2-X3-X4-X5-X6-X7 (I).

CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY

The application claims the benefit of Taiwan Application No. 104119897,filed on Jun. 22, 2015, at the Taiwan Intellectual Property Office, thedisclosures of which are incorporated herein in their entirety byreference.

FIELD OF THE INVENTION

The invention is related to a medical, dental or toilet preparation. Inparticular, it is related to the preparation of one kind of peptidefragments, which can be used in patients suffering from a disseminatedmycobacterial infection.

BACKGROUND OF THE INVENTION

Anticytokine autoantibodies (ACADs) are increasingly recognized, andplaying an important role in the pathogenesis of infectious andautoimmune diseases.

Clinically, the pathogenic mycobacterial species can cause tuberculosis,Hansen's disease, leprosy, pulmonary disease, lymphadenitis and skindisease. In view of mycobacterial immunity, IFN_(r) plays an importantrole and is mainly produced by T and NK cells when stimulated withmicrobial products.

Genetic defects in the IFN_(r)/IL-12 pathway cause Mendeliansusceptibility to mycobacterial diseases (MSMDs) in young patients withdisseminated mycobacterial infections.

On the other hand, interference with the IFN_(r) signaling by thepresence of anti-IFN_(r) AutoAbs is the major etiology that explains theoccurrence of severe disseminated mycobacterial infections in adultswithout obvious immunologic defects, in particular for patients from theSoutheast Asia.

The similarity of clinical susceptibility to MSMDs strongly suggeststhat AutoAbs against IFN_(r) were the cause rather than a consequence ofmycobacterial infection.

The mechanism of the production of anti-IFN_(r) AutoAbs also remainsunclear. Restriction of the disease in Southeast Asian populationsuggest that a particular genetic factor and mechanism are involved.

According to a previous study, HLA class II molecules DRB1*16:02 andHLA-DQB1*05:02 are the two specific alleles strongly associated withthis disease, and the high frequency of this allele in Southeast Asiamight also explain the susceptibility of anti-IFN_(r) AutoAbs in thisparticular population.

MHC class II is present the particular peptides to CD4⁺ T cells toinduce an adaptive immune response and is a strong genetic factorassociated with autoimmune diseases. It seems that particular pathogenicpeptide fragments present in these particular HLA alleles are involvedin the production of anti-IFN_(r) AutoAbs.

Various hypotheses have been proposed to explain the production of thesepathogenic AutoAbs. Molecular mimicry theory states that exo-antigen canmimic self-antigen and induces the formation of AutoAbs. This theory,molecular mimicry, has been documented in various autoimmune diseases,including multiple sclerosis (MS), ankylosing spondylitis, Graves'disease, diabetes mellitus, and systemic lupus erythematosus (SLE).

In the case of MS and SLE, the disease pathogenesis has been linked tosome viruses, such as the Epstein-Barr virus (EBV), for their homologousamino acid sequences with human antigenic structures in the centralnerve system or lupus autoantigens, such as Sm B.

These findings suggest that molecular mimicry plays a major role in thepathogenesis of certain diseases. Despite advances in the genomictechnologies for autoimmunity, the precise mechanism for the pathogenicAutoAbs formation is still unclear.

SUMMARY OF THE INVENTION

Autoantibodies (AutoAbs) against IFN_(r) is an emerging medical issueand linked to disseminated mycobacterial infections and otheropportunistic infections in the Southeast Asia. The origin of theseAutoAbs is unclear; however, the majority of affected patients sharespecific HLA class II alleles and this observation suggests that acommon mechanism in the production of AutoAbs may exist. Herein, theinventor characterized the anti-IFN_(r) AutoAbs from patients and foundthese AutoAbs recognized a major epitope (P₁₂₁₋₁₃₁) in the C-terminal ofIFN_(r). The region was known to be critical for IFN_(r) receptoractivation, and the inventor also demonstrated that AutoAbs to thisepitope had a neutralizing activity. This epitope was 100% homologous tothe Aspergillus Noc2 and anti-IFN_(r) AutoAbs from patients could reactwith this epitope and Aspergillus Noc2. In vivo study, rats immunizedwith Aspergillus Noc2 developed antibodies against human IFN_(r) andvice versa.

In addition, the inventor generated an epitope Erase IFN_(r)(EE-IFN_(r)) which has lower affinity recognized by anti-IFN_(r) AutoAbsdue to a modified major epitope region and it could activate the IFN_(r)downstream signaling pathway ex vivo even in the presence ofanti-IFN_(r) AutoAbs.

It was found that anti-IFN_(r) ⋅AutoAbs from different patientsrecognized a specific region, SPAAKTGKRK (SEQ ID NO: 14), in theC-terminal of IFN_(r) and these antibodies had a neutralizing activityon IFN_(r).

A high homologous peptide sequence in this region (KTGKRKR (SEQ ID NO:36)) was found in Aspergillus Noc2 and also recognized by theanti-IFN_(r) AutoAbs.

After immunization with Aspergillus Noc2, antibodies against humanIFN_(r) ⋅ were formed in the test rats.

Furthermore, the inventor used a mouse homologous region to replace thecritical anti-IFN_(r) ⋅AutoAb-recognized epitope in human IFN_(r)□ andgenerated a new recombinant protein, epitope Erased IFN_(r)⋅(EE-IFN_(r)).

This recombinant protein could induce the activation of the IFN_(r)receptor, even in the presence of anti-IFN_(r) ⋅AutoAbs ex vivo.

Taken together, the results suggest that anti-IFN_(r) ⋅AutoAbs may beinduced by the means of molecular mimicry, and structural modificationof IFN_(r) ⋅ can bypass the blocking activity of anti-IFN_(r) ⋅AutoAbs.

In accordance with one aspect of the present invention, a method forevaluating an efficacy of an isolated recombinant human interferon gamma(hIFN_(r)) for regulating a peripheral blood mononuclear cell (PBMC) isdisclosed. The method comprises the steps of: providing the PBMC from asubject with anti-interferon gamma autoantibodies; mixing the isolatedrecombinant human interferon gamma with the PBMC, wherein the isolatedrecombinant human interferon gamma contains a homologous substitute; andevaluating the efficacy of the isolated recombinant human interferongamma according to an expression level of a phosphorylation of signaltransducers and activators of transcription 1 (p-STAT1) generated by thePBMC.

In accordance with another aspect of the present invention, a method forevaluating an efficacy of an isolated recombinant cytokine forregulating a peripheral blood mononuclear cell (PBMC) is disclosed. Themethod comprises steps of: providing the PBMC from a subject with ananticytokine autoantibody; mixing the isolated recombinant cytokine withthe PBMC, wherein the isolated recombinant cytokine contains ahomologous substitute; and evaluating the efficacy of the isolatedrecombinant cytokine according to an expression level of aninterleukin-12 (IL-12) generated by the PBMC.

In accordance with another aspect of the present invention, arecombinant protein is disclosed. The recombinant protein comprises: ahuman interferon gamma having a sequence replaced with a peptide of“Leu-Pro-Glu-Ser-Ser-Leu-Arg” (SEQ NO: 1), wherein the recombinantprotein is used to activate a receptor of an interferon gamma (IFN_(r))and is free from neutralization by an autoantibody of the interferongamma of a subject suffering from a disseminated mycobacterialinfection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1h illustrate AutoAbs to IFN_(r) recognized C-terminal regionof IFN_(r) according to the embodiment of the present invention;

FIGS. 2a-2e illustrate epitope Erase IFN_(r) according to the embodimentof the present invention;

FIGS. 3a-3g demonstrate molecular mimicry;

FIGS. 4a-4h illustrate epitope Erase IFN_(r) and the applicationtherefor according to the embodiment of the present invention;

FIG. 5 illustrates IFN_(r) direct ELIAS used to detect AutoAbs againstIFN_(r);

FIGS. 6a-6b illustrate that IL-12 production levels and IFN_(r)production levels were measured by ELISA;

FIG. 7 illustrates the result of epitope mapping;

FIG. 8 illustrates the result of Western blot analysis conducted onvarious recombinant interferons;

FIG. 9 illustrates the results of probing plasma sampled from IFN_(r)AutoAbs patients and healthy controls;

FIG. 10 illustrates the results of protein mapping;

FIGS. 11a-11b illustrate the results of plasma interacting with hIFN_(r)and mIFN_(r);

FIG. 12 illustrates the results of Western blot analysis conducted ondifferent various recombinant Noc2 proteins; and

FIGS. 13a-13b illustrate the results of p-STAT-1 expression levels andHLA-DR expression levels.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more specifically withreference to the following embodiments. It is to be noted that thefollowing descriptions of preferred embodiments of this invention arepresented herein for the purposes of illustration and description only;they are not intended to be exhaustive or to be limited to the preciseform disclosed.

[Patients and Definitions]

All participants were adults (age >20 years) and were followed regularlyat medical centers in Taiwan. Disseminated mycobacterial infection wasdiagnosed if patients presented 2 or more noncontiguous places andpositive blood or BM cultures. The diagnosis of pulmonary NTM infectionwas based on the criteria proposed by the American Thoracic Society andInfectious Diseases Society of America. The study was approved by theInstitutional Review Board of China Medical University Hospital(DMR98-IRB-261) and Chang Gung Memorial Hospital (103-2861C), andinformed written consent was obtained from all of the patients inaccordance with the Declaration of Helsinki.

[Reagents]

The inventor used the following monoclonal antibodies: anti-humanIFN_(r) (clone EPR1108, Abcam), anti-V5 tag (clone SV5-Pk1, Abcam), FITCmouse anti-human CD14 (clone M5E2, BD Pharmingen), PE mouse anti-humanHuman Leukocyte Antigen-antigen D Related (HLA-DR)(clone G46-6, BDPharmingen) and PE Mouse Anti-Stat1 (pY701) (clone 4a, BD Phosflow).

[Detection and Titration of IFN_(r) Neutralizing Autoantibodies]

Blood plasma from patients and donors was serially diluted (10⁻¹ to10⁻⁶) and incubated with recombinant human IFN_(r) at a finalconcentration of 200 pg/mL for 1 hour at 37° C. The IFN_(r) level wasmeasured with a human IFN_(r) ELISA kit from BD Biosciences according tothe manufacturer's recommendations. All experiments were performed induplicate.

[Peptide Synthesis]

All peptides were synthesized by Kelowna International Scientific Inc.,Taiwan. The peptides were lyophilized, and the purity and mass wereassessed by high-performance liquid chromatography and massspectrometry, respectively.

[Epitope Mapping]

Clear polystyrene 96-well flat bottom plates (Nunc) were coated with 100μL of peptide solution (5 μg/mL) with b-carbonate buffer (pH 9.6) at 4°C. overnight. After blocking the remaining binding sites with 5% humannormal serum albumin (Aventis) in PBS, the plates were incubated at 37°C. for 1 hour. Afterward, the plates were washed 3 times with 0.5% Tween20 in PBS.

Blood plasma samples were added at a 1:100 dilution, in PBS containing5% human normal serum albumin. Following incubation at room temperaturefor 2 hours and being washed 3 times, goat anti-human IgG Fc_(r)fragment specific conjugated with alkaline phosphate (JacksonImmunoResearch Laboratories), was added at a 1:5000 dilution, and theplates were kept for 1 hour at room temperature. Finally, after beingwashed 5 times, 100 μL of p-Nitrophenyl Phosphate (pNPP) was added andthe reaction was read at 405 nm by a VICTOR X3 Multilabel Plate Reader(PerkinElmer) after 30 minutes of incubation at 37° C.

[Competition Enzyme-Linked Immunosorbent Assay (ELISA)]

To evaluate the specificity of the reaction, a competition ELISAexperiment was performed. Each plasma sample (1:100 dilutions) waspre-incubated with different concentrations (5-30 μg/mL) of thecompetition peptides overnight at 4° C. Subsequently, all plasma anddifferent concentrations competition peptides mixture samples wereexamined following the epitope mapping protocol. Peptide 6 was coatedwith b-carbonate buffer (pH 9.6) at 4° C. overnight. After blocking at37° C. for 1 hour, all pre-incubated plasma and different concentrationscompetition peptides mixture samples were added in duplicate. Following2 hours of room temperature incubation, goat anti-human IgG Fc_(r)fragment specific conjugated with alkaline phosphate, was added.Finally, 100 μL of pNPP was added and the reaction was read at 405 nm bya VICTOR X3 Multilabel Plate Reader (PerkinElmer) after 30 minutes ofincubation at 37° C.

[Prediction of B Cell Epitopes]

The prediction of B cell epitopes was carried out using BepiPred Linearepitope Prediction and Emini Surface Accessibility Prediction. Thesoftware takes a single sequence in FASTA format as an input. BepiPredpredicts the location of linear B-cell epitopes using a combination of ahidden Markov model and a propensity scale method. In Emini SurfaceAccessibility Prediction, the calculation was based on surfaceaccessibility scale of a product instead of an addition within thewindow. The accessibility profile was obtained using a special formula.

[Generation of Recombinant Human IFN_(r)]

The inventor amplified the IFN_(r)⋅ from base 196 to 624 of the openreading frame (ORF) by polymerase chain reaction (PCR) and cloned itinto pMT/BiP/V5-His (Invitrogen) for Drosophila Schneider 2 (S2) cellsexpression. Recombinant IFN_(r)⋅ was further purified by V5 taggedprotein purification kit (MBL). The mutated recombinant IFN_(r)⋅ wasperformed by QuickChange Site-Directed Mutagenesis kit (Stratagene). Theinventor confirmed the sequences and cloning sites of all constructs aswell as the size and immunoreactivity of the recombinant IFN_(r)⋅ byWestern Blot.

[Generation of Recombinant Aspergillus Noc2]

The inventor amplified the Noc2 from base 1 to 2361 of the ORF by PCRand cloned it into pMT/BiP/V5-His (Invitrogen) for S2 cells expression.Recombinant Noc2 were further purified by a V5 tagged proteinpurification kit (MBL). Then, the sequences and cloning sites of allconstructs were confirmed as well as the size and immunoreactivity ofrecombinant Noc2 by Western Blot.

[p-STAT1 Intracellular Stain]

A total of 10⁶ PBMCs in 200 μL RPMI-1640 with 10% FBS and 1%penicillin/streptomycin was used. Cells were then stimulated with 500 IUIFN_(r), IFN_(r) _(_)1-131 or EE-IFN_(r)□ in 160 μL RPMI-1640pre-incubated with 40 μL of normal plasma or patient plasma for 10 minat room temperature. After 30 minutes of stimulation in a 37° C.incubator, monocytes were identified by FITC-CD14 (BD Pharmingen)surface staining. Fixed by adding 250 μL of FASC lysing solution (BDPharmingen) and incubated at room temperature for 15 minutes in thedark, followed by two washes with PBS. To permeabilize the cells, 500 μlof ice-cold absolute methanol was added to each tube and incubated for15 minutes on ice in the dark, followed by two washes with PBS. Next,PE-phospho-STAT1 (pY701) antibody (BD Pharmingen) was added, followed by30 minutes incubation on ice in the dark. Again, cells were washed with2 mL of PBS before resuspension in the 500 μL of PBS. Data werecollected with a FACSVerse flow cytometer (BD Biosciences) and analyzedusing FACSuite software (BD Biosciences).

[Immunoblotting]

Recombinant IFN_(r)□ (R&D Systems) and different truncated forms ofrecombinant IFN_(r)□ were subjected to SDS-PAGE using a 10% gel underreducing conditions at 120 V for 2 hours. The proteins were transferredto a PVDF membrane (Invitrogen Life Technologies) at 250 mA for 2 hours.The membrane was blocked in 5% human normal serum albumin (Aventis) inTBS with 0.1% Tween 20 overnight at 4° C. The membrane was incubatedwith a 1/100 dilution of patient or control blood plasma for 3 hours atroom temperature. After being washed three times, the membrane wasincubated in a 1/10,000 dilution of mouse anti-human IgG conjugated withhorseradish peroxidase (Jackson ImmunoResearch Laboratories) for 1 hourat room temperature. After being washed three times, the membrane wasdeveloped with ECL (Merck Millipore).

[HLA-DR Expression]

A total of 10⁶ PBMCs in 200 μL RPMI-1640 with 10% FBS and 1%penicillin/streptomycin was used. Cells were then stimulated with 500 IUIFN_(r), IFN_(r) _(_)1-131 or EE-IFN_(r)⋅ in 160 μL RPMI-1640pre-incubated with 40 μL of normal blood plasma or patient blood plasmafor 10 min at room temperature. After 24 hours of stimulation in a 37°C. incubator, monocytes were identified by FITC-CD14 (BD Pharmingen)surface staining. Next, PE-HLA-DR antibody (BD Pharmingen) was addedfollowed by 30 minutes of incubation on ice in the dark. Cells wereagain washed with 2 ml of PBS before resuspension in 500 μl of PBS. Datawere collected with a FACSVerse flow cytometer (BD Biosciences) andanalyzed using FACSuite software (BD Biosciences).

Immunization with Noc2 or IFN_(r)⋅ Peptide

The inventor immunized WKY/NcrlNarl rats with 0.25 mg Noc2 or IFN_(r)□peptide (Noc2: CTPKTGKRKRSEQ (SEQ NO: 31); IFN_(r): CAAKTGKRKRSQM (SEQNO: 32)) conjugated with ovalbumin (OVA) and gave a booster dose everytwo weeks after the initial injection (day 0). After 70 days, theinventor checked the antibody production level by dot blot or ELISA ateach injection. All rats were sacrificed ten days after the tenthinjection (day 136) and whole blood was collected. After collection ofthe whole blood, the blood was allowed to clot by leaving it undisturbedat room temperature 15 minutes. Clots were removed by centrifuging at1500 g for 10 minutes in a refrigerated centrifuge. Sera were aliquotedand stored at −20° C. for further use.

[Experimental Data]

Please refer to FIGS. 1a-1h , which are related to AutoAbs to IFN_(r)recognized C-terminal region of IFN_(r).

FIG. 1a demonstrates amino acid sequences of human IFN_(r)⋅30-meroverlapping synthetic peptides.

FIG. 1b (1)-FIG. 1b (6) demonstrate epitope mapping for determiningserum binding affinity of the synthetic peptide to IFN_(r), representedas the mean optical density (OD) at 405 nm; plasma sample from IFN_(r)AutoAbs patients (n=15), Mycobacteria infected patients (n=2) andhealthy controls (n=6).

FIG. 1c (1)-FIG. 1c (2) demonstrate the inhibition using ELISA, whereineach plasma sample from IFN_(r)⋅AutoAbs patients was pre-incubated withdifferent concentrations of a control peptide or peptide 6, and then allplasma dilutions were examined by ELISA for reactivity against peptide6.

FIG. 1d demonstrates a schematic diagram of IFN_(r), and the predictionscores for linear B cell epitopes and surface acceptability regions.Each green bar represents a predicted B cell epitope, and each red barrepresents a predicted surface acceptable region. The prediction scoresrepresent the average scores for all amino acids within the region withprediction values above the cut-offs chosen for significance. The barcolor intensities are proportional to the prediction scores found.

FIG. 1e demonstrates a three-dimensional projection of the structure ofIFN_(r). The epitope, amino acids 121-131 of IFN_(r)⋅ are located at theC-terminal and extend into the solvent.

FIG. 1f demonstrates a graphical representation of the different lengthof truncated recombinant IFN_(r) proteins.

FIG. 1g (1)-FIG. 1g (6) demonstrate protein mapping used to determineplasma binding affinity of the recombinant proteins to IFN_(r),represented as the mean optical density (OD) at 405 nm; plasma samplefrom IFN_(r)⋅AutoAbs patients (n=16), Mycobacteria infected patients(n=2) and healthy controls (n=3).

FIG. 1h demonstrates a Western blot showing binding ability of bloodplasma from IFN_(r)⋅AutoAbs patients (n=4, P1-4) and healthy controls(n=2, C1-2) to different truncated forms of IFN_(r).

Please refer to Table 1, which demonstrates multiple amino acid sequencealignments of IFN_(r) in different species and EE-IFN_(r) (SEQ NO:15-21). The conserved residues in the other species are boxed. Thesequence of EE-IFN_(r) protein, which has substituted human P₁₂₁₋₁₂₇Ser-Pro-Ala-Ala-Lys-Thr-Gly (SPAAKTG) (SEQ ID NO: 39) with homologousmurine Leu-Pro-Glu-Ser-Ser-Leu-Arg (LPESSLR) addressing SEQ NO: 1 are inbold.

TABLE 1

In other embodiments, it is possible to apply homologous substituteswith various peptide lengths to replace the sequence in human P₁₂₁₋₁₂₇.For example, a homologous substitute comprises at least one amino acidcapable of fulfilling at least one element loss in the epitope. Foranother example, a mutation procedure is applied in at least one elementin the epitope.

FIG. 2a (1)-FIG. 2a (2) demonstrate the affinity of anti-IFN_(r) AutoAbsto IFN_(r) _(_)1-131 and EE-IFN_(r)□□ by ELISA. Anti-IFN_(r)⋅AutoAbsfrom 5 patients' blood plasma was bound to IFN_(r) _(_)1-131 in a dosedependent manner but not to EE-IFN_(r)⋅ represented as the mean opticaldensity (OD) at 405 nm.

FIG. 2b demonstrates a Western blot analysis showing the binding ofblood plasma from Anti-IFN_(r)□AutoAbs patients (n=4, P1-4) and healthycontrols (n=2, C1-2) to IFN_(r)1-131 and EE-IFN_(r). Plasma from 3healthy donors had no response to either IFN_(r)1-131 or EE-IFN_(r).

FIGS. 2c-2e demonstrate PBMCs from healthy controls that werepre-incubated with 20% Ctrl plasma (n=6) or anti-IFN_(r) AutoAbspatients' plasma (n=6) and treated with WT IFN_(r), IFN_(r) _(_)1-131 orEE-IFN_(r)□□ After being gated on the CD14⁺, p-STAT-1 levels weredetermined by flow cytometry with the p-STAT-1 monoclonal antibody.Representative histograms (FIG. 2c (1)-FIG. 2c (2)) and MFI (medianfluorescence intensity) (FIG. 2d ) are shown.

FIG. 2e demonstrates that PBMCs from healthy controls were pre-incubatedwith 20% Ctrl plasma (n=4) or anti-IFN_(r) AutoAbs patients' bloodplasma (n=9) and stimulated with BCG BCG plus WT IFN_(r), BCG plusIFN_(r) _(_)1431 or BCG plus EE-IFN_(r)⋅⋅IL12p40 levels in thesupernatant were measured by ELISA.

Table 2 demonstrates homologies in hIFN_(r)⋅ peptides P₁₂₅₋₁₃₃ toAspergillus spp. Noc2 proteins. Amino acid numbers correspond to thoseof proteins published by The National Center for BiotechnologyInformation (NCBI). AA is an abbreviation for number of amino acids. Andthe proteins were sampled from Homo sapiens (SEQ NO: 22), Aspergillusterreus (SEQ NO: 23), Aspergillus fumigatus (SEQ NO: 24), Aspergillusflavus (SEQ NO: 25), Aspergillus nidulans (SEQ NO: 26), Aspergillusniger (SEQ NO: 27), Cryptococcus neoformans (SEQ NO: 28), Mycobacteriumintracellulare (SEQ NO: 29) and Clostridium sp. MSTE9 (SEQ NO: 30).

TABLE 2  Accession  Protein Species AA AA Positive code IFNγ P₁₂₅₋₁₃₃Homo sapiens 125 AAKTGKRKRSQML 133 — GI:56786138 (SEQ ID NO: 22)Ribosome assembly  Aspergillus  105 TPKTGKRKRSEQQ 113 9/9 (100%)GI:115397505 protein Noc2 terreus (SEQ ID NO: 23) Ribosome assembly Aspergillus  100 TPKITGKRKRSEEQ 108 8/9 (89%)  GI:159131733 protein Noc2fumigatus (SEQ ID NO: 24) Ribosome assembly  Aspergillus  102TPKTGKRKRTEEQ 110 8/9 (89%)  GI:238483989 protein Noc2 flavus(SEQ ID NO: 25) Ribosome assembly  Aspergillus  103 SPKIGKRKRSETQ 1118/9 (89%)  GI:67517347 protein Noc2 nidulans (SEQ ID NO: 26)Ribosome assembly  Aspergillus  102 TPKIGKRKRSDEQ 110 7/9 (78%) GI:317025642 protein Noc2 niger (SEQ ID NO: 27) Hypothetical Cryptococcus  623 LGSTGKRKRSSMG 631 7/9 (78%)  GI:405119493protein CNAG_05000 neoformans (SEQ ID NO: 28) Hypothetical Mycobacterium  124 NPAGGKRKRSQ-- 132 7/9 (78%)  GI:379753578protein OCO_15660 intracellulare (SEQ ID NO: 29) Diguanylate Clostridium  14 KEQSKKRKRSQMT 22 6/9 (67%)  GI:496353461 cyclasesp. MSTE9 (SEQ ID NO: 30)

FIG. 3a (1)-FIG. 3a (2) demonstrate epitope cross reaction mapping usedto determine blood plasma binding affinity of the synthetic peptide toAspergillus Noc2, represented as the mean optical density (OD) at 405nm; blood plasma sample from IFN_(r) AutoAbs patients (n=9),Mycobacteria infected patients (n=3) and healthy controls (n=3).

FIG. 3b (1)-FIG. 3b (2) demonstrate inhibition of ELISA, where eachplasma sample from IFN_(r) AutoAbs patients (n=7) was pre-incubated withdifferent concentrations of control peptide or Aspergillus Noc2 peptide,then all plasma dilutions were examined by ELISA for reactivity againstpeptide 6.

FIG. 3c demonstrates a Western blot analysis showing binding of plasmaform Anti-IFN_(r)⋅AutoAbs patients (n=4, P1-4) and healthy controls(n=2, C1-2) truncated Aspergillus Terreus (Amino Acid: 1-165) andAspergillus Fumigatus (Amino Acid: 1-162) recombinant Noc2 protein.

FIG. 3d (1)-FIG. 3d (4) demonstrate immunization with Noc2 peptideinduced antibodies to IFN_(r). Blood sera from pre-immunized rats (n=6),Noc2 peptide-immunized rats (n=4) and Peptide 6-immunized rats (n=2)were mapped to control peptide, Peptide 6 and Noc2 peptide fromAspergillus terreus or Aspergillus fumigatus. This is represented as themean optical density (OD) at 405 nm.

FIG. 3e demonstrates blood sera from pre-immunized rats (n=2), Noc2peptide-immunized rats (n=4) and Peptide 6-immunized rats (n=2) wereserially diluted and incubated with 200 pg/mL of human IFN_(r). Serafrom pre-immunized rats showed no blocking activities for the detectionof hIFN_(r). However, blood sera from Noc2 peptide-immunized rats (n=4)and Peptide 6-immunized rats (n=2) at dilutions of up to 1/10² inhibitedthe detection of hIFN_(r).

FIGS. 3f-3g demonstrate that THP1 cells were pre-incubated with 50%blood sera from pre-immunized rats (n=2), Noc2 peptide-immunized rats(n=4) or Peptide 6-immunized rats (n=2) and treated with WTIFN_(r)⋅⋅p-STAT-1 levels were determined by flow cytometry with thep-STAT-1 monoclonal antibody, MFI (median fluorescence intensity) areshown in FIG. 3f HLA-DR expression levels were determined by flowcytometry with the HLA-DR monoclonal antibody, and MFI are shown in FIG.3 g.

FIGS. 4a-4b demonstrate that PBMCs from healthy controls (n=5) oranti-IFN_(r) AutoAbs patients (n=6) were pre-incubated with 20%autologous plasma and treated with WT IFN_(r), IFN_(r)1-131 orEE-IFN_(r). After being gated on the CD14⁺, p-STAT-1 levels weredetermined by flow cytometry with the p-STAT-1 monoclonal antibody.Representative histograms (FIG. 4a (1)-FIG. 4a (2)) and MFI (medianfluorescence intensity) (FIG. 4b ) are shown.

FIG. 4c demonstrates that PBMCs from healthy controls (n=3) oranti-IFN_(r) AutoAbs patients (n=6) were pre-incubated with 20%autologous blood plasma and stimulated with BCG BCG plus WT IFN_(r), BCGplus IFN_(r) _(_)1-131 or BCG plus EE-IFN_(r)⋅⋅IL12p40 levels in thesupernatant were measured by ELISA.

FIGS. 4d-4e demonstrate that PBMCs from healthy controls (n=5) oranti-IFN_(r) AutoAbs patients (n=4) were pre-incubated with 20%autologous plasma and treated with WT IFN_(r), IFN_(r) _(_)1-131 orEE-IFN_(r). After being gated on the CD14⁺, HLA-DR expression levelswere determined by flow cytometry with the HLA-DR monoclonal antibody.Representative histograms (FIG. 4d (1)-FIG. 4d (2)) and MFI (medianfluorescence intensity) (FIG. 4e ) are shown.

FIGS. 4f-4g demonstrate that whole blood from healthy controls (n=4) oranti-IFN_(r) AutoAbs patients (n=6) was treated with WT IFN_(r), IFN_(r)_(_)1-131 or EE-IFN_(r)⋅ ⋅ After being gated on the CD14⁺, p-STAT-1levels were determined by flow cytometry with the p-STAT-1 monoclonalantibody. Representative histograms (FIG. 4f (1)-FIG. 4f (2)) and MFI(median fluorescence intensity) (FIG. 4g ) are shown.

FIG. 4h demonstrates that whole blood from healthy controls (n=4) oranti-IFN_(r) AutoAbs patients (n=5) was stimulated with BCG BCG plus WTIFN_(r), BCG plus IFN_(r) _(_)1-131 or BCG plus EE-IFN_(r)⋅IL12p40levels in the supernatant were measured by ELISA.

FIG. 5 demonstrates that direct ELIAS was used to detect AutoAbs againstIFN_(r). Represented as the mean optical density (OD) at 405 nm; bloodplasma samples from IFN_(r) AutoAbs patients (n=2), and healthy controls(n=65) are shown.

FIG. 6a demonstrates that IL-12 production levels were measured by ELISAafter non-activated, BCG activated or BCG plus IFN_(r) activated inwhole blood from three groups individuals were tested. IFN_(r) AutoAbspatients (n=3), healthy controls without AutoAbs (n=7), and healthycontrols with non-neutralizing IFN_(r)⋅AutoAbs (n=4) are shown.

FIG. 6b demonstrates that IFN_(r) production levels were measured byELISA after non-activated, BCG activated or BCG plus IL-12 activated inwhole blood from three groups individuals were tested. IFN_(r) AutoAbspatients (n=3), healthy controls without AutoAbs (n=7), and healthycontrols with non-neutralizing IFN_(r)⋅AutoAbs (n=4) are shown.

FIG. 7 demonstrates epitope mapping used to determine plasma bindingaffinity of the synthetic peptide to IFN_(r), represented as the meanoptical density (OD) at 405 nm including plasma samples from IFN_(r)AutoAbs patients (n=3, P1-3), healthy controls without AutoAbs (n=2,C1-2), and healthy controls with non-neutralizing IFN_(r)⋅AutoAbs (n=5,N1-5).

FIG. 8 demonstrates a Western blot analysis showing the expression ofdifferent forms of recombinant IFN_(r) by a V5-tag antibody.

FIG. 9 demonstrates blood plasma sampled from IFN_(r)⋅AutoAbs patients(n=5, P1-5) and healthy controls (n=2, C1-2) were probes for vector,IFN_(r) _(_)1-50, IFN_(r) _(_)1-70, IFN_(r) _(_)68-121, IFN_(r)_(_)1-121, IFN_(r) _(_)1-131, and IFN_(r) _(_)1-139 recombinantproteins.

FIG. 10 demonstrates protein mapping used to determine blood plasmabinding affinity of the recombinant proteins to IFN_(r), represented asthe mean optical density (OD) at 405 nm including blood plasma samplesfrom healthy controls without AutoAbs (n=3, C1-3), and healthy controlswith non-neutralizing IFN_(r)⋅AutoAbs (n=5, N1-5).

FIGS. 11a-11b demonstrate that seven patients' blood plasma was seriallydiluted and incubated with 200 pg/mL of human IFN_(r) (hIFN_(r)) ormurine IFN_(r) (mIFN_(r)). The blood plasma from healthy donors showedno blocking activities for the detection of hIFN_(r) or mIFN_(r).However, plasma from patients with AutoAbs against IFN_(r) at dilutionsof up to 1/10⁴ inhibited the detection of hIFN_(r) but showed noblocking activity for the detection of mIFN_(r).

FIG. 12 demonstrates a Western blot analysis showing the expression ofdifferent forms of recombinant Noc2 by V5-tag antibody.

FIGS. 13a-13b demonstrate that THP1 cells were pre-incubated with 50%Sera from pre-immunized rats (n=2), Noc2 peptide-immunized rats (n=4) orPeptide 6-immunized rats (n=2) and treated with WT IFNγ p-STAT-1, andlevels were determined by flow cytometry with the p-STAT-1 monoclonalantibody, wherein representative histograms (FIG. 13a (1)-FIG. 13a (2))are shown. HLA-DR expression levels were determined by flow cytometrywith the HLA-DR monoclonal antibody, wherein representative histograms(FIG. 13b (1)-FIG. 13b (2)) are shown.

[Embodiment I] Anti-IFN_(r) Autoantibodies Recognize a C-Terminal Regionin IFN_(r) Peptide⋅

The inventor tried to identify the epitope recognized by anti-IFN_(r)AutoAbs isolated from patients by peptide scan: Six 30-mer peptides,overlapping by a 7 or 8 amino acids and covering the entire codingsequence of human IFN_(r), were synthesized, and the amino acidssequences of peptides addressing SEQ NOS: 2 to 7 were listed (FIG. 1a ).Epitope mapping assay was performed using plasma from three differentpatient groups: anti-IFN_(r) AutoAbs patients with disseminatedmycobacterial infections (n=15, group-1), patients with mycobacterialinfections without anti-IFN_(r) AutoAbs (n=2, group-2), and healthycontrols (n=6, group-3). It was found that only peptide 6 was recognizedin the blood plasma isolated from the 12 patients in group-1 (FIG. 1b(1)-FIG. 1b (6)). In contrast, no binding activity to these six peptideswas observed in other two patient groups and this observation suggeststhat recognition of the peptide 6 was not due to a cross-reaction ofanti-mycobacterial antibodies. Next, competition ELISA was used toconfirm the specificity of the anti-IFN_(r) AutoAbs to the peptide 6.Blood plasma from group-1 patients was pre-incubated with peptide 6,which led to competition for the binding with coated peptide 6 in a dosedependent manner, and no competition was observed when the controlpeptides (peptide 1-5) were used as competitors (FIG. 1c (1)-FIG. 1c(2)). These data suggest that the anti-IFN_(r) AutoAbs from group-1patients was recognized by one major region in the peptide 6 (P₁₁₄₋₁₄₃).

In contrast to neutralizing AutoAbs to IFN_(r)⋅ in patients withmycobacterial infections, non-neutralizing AutoAbs against IFN_(r) hadbeen reported in healthy individuals. Five donors were found withAutoAbs against IFN_(r) out of 65 healthy controls by ELISA (FIG. 5).AutoAbs from these five healthy individuals showed a non-neutralizingeffect on IFN_(r) in terms of induction of IL-12 production. In contrastto neutralizing IFN_(r)⋅AutoAbs from patients, non-neutralizing AutoAbsfrom healthy donors did not recognize peptide 6 (FIG. 8). These dataindicate that the IFNγ AutoAbs from patients with disseminatedmycobacterial infections, but not non-neutralizing IFNγ AutoAbs,recognized a major region on peptide 6.

[Embodiment II] Predictions of B-Cell Epitopes Using Computer Modeling

For further confirmation of the precise region recognized byanti-IFN_(r) AutoAbs, the inventor scanned the primary sequence ofIFN_(r) for possible B-cell linear epitopes and possible surfaceacceptability in silico. It was found several candidate epitopesaddressing SEQ NO: 8-14 in the full length IFN_(r)⋅(FIG. 1d ).Considering the higher prediction scores for screening features andlocated in the peptide 6, the epitope P₁₂₁₋₁₃₁ SPAAKTGKRKR (SEQ ID NO:33) was predicted in B-cell epitope prediction algorithms and P₁₂₆₋₁₃₁TGKRKR (SEQ ID NO: 37) was predicted in Surface Acceptability predictionalgorithms. These results were consistent with peptide epitope mappingdata. The critical epitope P₁₂₁₋₁₃₁ SPAAKTGKRKR (SEQ ID NO: 33) that weidentified is located on an unfolded sequence in the C-terminal region.It extends into the solvent and does not strongly interact with theremainder of the molecules (FIG. 1e ).

[Embodiment III] Anti-IFN_(r) Autoantibodies Recognize a C-TerminalRegion in IFN_(r)⋅ Protein

In order to investigate whether anti-IFN_(r) AutoAbs from group-1patients recognized the same region in the native IFN_(r) protein, wegenerated various truncated IFN_(r) by Schneider 2 cell expressionsystem (FIG. 1f and FIG. 9). It was found that only full-length IFN_(r)(IFN_(r) _(_)1-144) and deletion clone IFN_(r) _(_)1-131 were recognizedby blood plasma isolated from the group-1 patients (FIG. 1g (1)-FIG. 1g(6)). In contrast, blood plasma isolated from the group-2 and group-3patients did not recognize all the recombinant IFN_(r) tested. InWestern blot and dot blot assays, we also observed similar phenomenathat only full-length IFN_(r) _(_)1-144 (data not shown) and deletionclone IFN_(r) _(_)1-139 and IFN_(r) _(_)1-131 were recognized byanti-IFN_(r) AutoAbs, but not other truncates IFN_(r)⋅(FIGS. 1h and 10).These data suggest that a major anti-IFN_(r)□AutoAbs-recognized B-cellepitope, P₁₂₁₋₁₃₁ (SPAAKTGKRKR) (SEQ NO: 33) was located at theC-terminal of IFN_(r).

[Embodiment IV] EE-IFNγ

Inside the epitope region we identified, a.a. 128-131 (KRKR) (SEQ ID NO:38) (the last 4 resides in SEQ NO: 20) is crucial for the bioactivity ofIFN_(r)□ and conserved in most species.

Nevertheless, a.a. 121-127 is less conserved among different species(human: SPAAKTG (SEQ ID NO: 39) (the 6^(th)-12^(th) resides in SEQ NO:20); murine: LPESSLR (SEQ NO: 1)) (Table 1).

It had been observed that blood plasma from randomly selected patientswith anti-IFN_(r) AutoAbs did not show blocking activity on the murineIFN_(r) (FIG. 12), which suggests that SPAAKTG (the 6^(th)-12^(th)resides in SEQ NO: 20) is a necessary component for the binding ofanti-IFN_(r) AutoAbs. Previous studies demonstrated that the precisesequence in a.a. 121-127 was not critical for IFN_(r) functions, and thedeletion of some amino acids from the C-terminal of human IFN_(r)resulted in increasing bioactivity.

Following these observations, the inventor generated an EE-IFN_(r)protein by substituting the human P₁₂₁₋₁₂₇ SPAAKTG sequence (SEQ ID NO:39) (the 6^(th)-12^(th) resides in SEQ NO: 20) with murine LPESSLRsequence (SEQ NO: 1) in the IFN_(r) _(_)1-131 protein (Table 1).

In other embodiments, EE-IFNγ may be further combined withpharmaceutically acceptable excipients or carriers for clinical use.

The excipient in the present invention also refers to a pharmaceuticallyacceptable carrier or excipient, or a bio-available carrier orexcipient, including a solvent, dispersant, coating, antibacterial orantifungal agent, preservative or slow absorber, which is a propercompound used to prepare a formulation in the prior art. Usually such acarrier or excipient does not have any activity for treatments itself.And the compound disclosed in the present invention cooperating with apharmaceutically acceptable carrier or excipient is prepared as variousformulations, and will not result in adverse drug reactions, allergiesor other inappropriate responses after it is administered to animals orhumans. Thus the compound in the present invention, cooperating with apharmaceutically acceptable carrier or excipient, can be used in clinicsand humans.

“Effective dose” means a dose which is enough to improve or preventmedical symptoms or biological manifestation. The effective dose may bealso stated as a casting dose for use in diagnosis. Unless there isanother description in the specification, “active compound” and“pharmaceutically active compound” are substitutes for each other andrefer to a pharmaceutical, pharmacological or therapeutic substance aswell as other effective material.

Using ELISA, we observed that the affinity of anti-IFN_(r) AutoAbs toEE-IFN_(r) was markedly decreased in comparison with wild type (WT)IFN_(r) _(_)1-131 (FIG. 2a (1)-FIG. 2a (2)). With the aid of Westernblot, again, we observed a similar different affinity to anti-IFN_(r)AutoAbs between EE-IFN_(r) and WT IFN_(r) _(_)1-131 (FIG. 2b ). Thesedata suggest that the epitope of anti-IFN_(r) AutoAbs is located in a.a.121-131 and SPAAKTG was a necessary component for recognition.

[Embodiment V] Anti-IFN_(r) AutoAbs to Epitope P₁₂₁₋₁₃₁ haveNeutralizing Activity on IFN_(r)

The inventor aimed to know if anti-IFN_(r) AutoAbs to epitope P₁₂₁₋₁₃₁played a critical role in the neutralizing effect in an anti-IFN_(r)AutoAbs disease. A previous study showed that substitution of thehomologous murine sequence between residue 121 and residue 127 resultedin only a small decrease in biological activity. The biological activityof EE-IFN_(r) was tested by measuring the phosphorylation of signaltransducers and activators of transcription 1 (p-STAT1) signaling assayby flow cytometry and interleukin-12 (IL-12) production through ELISA.Up-regulation of the p-STAT1 signaling was observed in the controls'peripheral blood mononuclear cells (PBMCs) that were activated withrecombinant IFN_(r), WT IFN_(r) _(_)1-131, or EE-IFN_(r) in the presenceof 20% control blood plasma, which demonstrated that EE-IFN_(r) hadsimilar bioactivity with WT IFN_(r) _(_)1-131 in the p-STAT1 signaling(FIGS. 2c and 2d ). In pre-incubated with 20% blood plasma withanti-IFN_(r) AutoAbs from patients, only EE-IFN_(r), but not recombinantIFN_(r) or WT IFN_(r) _(_)1-131, could induce the phosphorylation ofSTAT1. These data strongly suggest that the anti-IFN_(r) AutoAbs hadneutralizing effects specific to epitope P₁₂₁₋₁₃₁. IFN_(r) couldincrease the synthesis and expression of HLA-DR in myeloid cells andother cells types. Therefore, the HLA-DR expression after IFN_(r)stimulation was measured. Similarly, up-regulation of HLA-DR expression,a process activated by IFN_(r), in THP1 cells was observed whilestimulated with EE-IFN_(r), even in the presence of anti-IFN_(r)AutoAbs. Similar to STAT1 and HLA-DR signaling, the elevation ofIL-12p40 expression couldn't be observed when pre-incubated with 20%patients' blood plasma and stimulated with BCG plus recombinant IFN_(r)or IFN_(r) _(_)1-131. In contrast, BCG plus EE-IFN_(r) did restoreIL-12p40 expression in the controls' PBMC pre-incubated with 20%patients' blood plasma (FIG. 2e ). These data indicated epitope P₁₂₁₋₁₃₁was the only or major binding site for anti-IFN_(r) AutoAbs in differentpatients, in terms of the blocking effect.

[Embodiment VI] IFN_(r) Epitope P₁₂₅₋₁₃₃ is Homologous to AspergillusConserved Protein

Recognition of a common epitope and shared HLA haplotypes among thepatients with Anti-IFN_(r) AutoAbs suggest that a common pathogenesismechanism of molecular mimicry might be involved. To test thispossibility, the inventor used P₁₂₁₋₁₃₅ a.a. sequence (SPAAKTGKRKRSQML)(SEQ NO: 34) to search for similar peptides in the human and microbedatabase. No similarity between this region and other human protein wasobserved; however it was found that the P₁₂₅₋₁₃₃ epitopes had 100%positives with amino acids 105-113 of the ribosome assembly protein Noc2(with eight of the nine amino acids identical) from Aspergillus terreus,and this region in Noc2 was conserved among most Aspergillus spp.available in the database (Table 2).

[Embodiment VII] IFN_(r) AutoAbs could Cross-React with Aspergillus Noc2

Next, the inventor investigated whether IFN_(r) AutoAbs from patientscould cross-react with Aspergillus Noc2. First, the antigenicity of thesynthetic Aspergillus Noc2 peptide (KKDVTPKTGKRKRSEQQKDE (SEQ NO: 35))was measured, which was evaluated by epitope mapping assay using bloodplasma isolated from patients with anti-IFN_(r) AutoAbs (FIG. 3a(1)-FIG. 3a (2)). This data show that the Aspergillus Noc2 peptide wasrecognized by blood plasma isolated from patients with anti-IFN_(r)AutoAbs, but it did not react with blood plasma from mycobacterialinfected patients without AutoAbs to IFN_(r) (FIG. 3a (1)-FIG. 3a (2)).The homogeneity between human IFN_(r) P₁₂₅₋₁₃₃ and Aspergillus Noc2 inthe conserved region P₁₀₅₋₁₁₃ was further assessed by competition ELISA.Samples isolated from patients with anti-IFN_(r) AutoAbs werepre-incubated with Aspergillus Noc2 peptide, and then reacted with thepeptide 6. The data show that the Aspergillus Noc2 peptide would competefor IFN_(r) AutoAbs binding with peptide 6 in a dose dependent mannerand no competition was observed when the control peptides were used ascompetitors (FIG. 3b (1)-FIG. 3b (2)). These data suggest that the humanIFN_(r) AutoAbs could cross-react with the Aspergillus Noc2 peptide.

[Embodiment VIII] Autoantibodies to IFN_(r) Recognized Aspergillus Noc2

To further investigate whether AutoAbs against IFN_(r) could recognizethe Aspergillus Noc2 in the protein level, we generated truncatedrecombinant Noc2 protein P₁₋₁₆₅ and P₁₋₁₆₂ from Aspergillus Terreus andAspergillus Fumigatus. A Western blot assay was performed to examine thecross-reaction of anti-IFN_(r) AutoAbs between human IFN_(r) andAspergillus Noc2. Utilizing blood plasma from patients with anti-IFN_(r)AutoAbs as the primary antibodies, it was found that anti-IFN_(r)AutoAbs recognize truncated recombinant Noc2 proteins from differentAspergillus species. In contrast, blood plasma from healthy controlscould not bind to truncated recombinant Noc2 proteins or WT IFN_(r)_(_)1-131 protein (FIG. 3c ). These data provide evidence thatanti-IFN_(r) AutoAbs from patients could cross-reacts with theAspergillus Noc2 protein.

[Embodiment IX] Noc2 Immunization Induces Antibodies to IFN_(r)

In order to investigate the immunogenicity to induce the anti-IFN_(r)AutoAbs to epitope P₁₂₁₋₁₃₁ in vivo, the inventor immunized four ratswith synthetic a.a. 103-114 Aspergillus Noc2 peptide conjugated withovalbumin (OVA). These rats developed anti-Noc2 antibodies thatcross-reacted with human IFN_(r) (FIG. 3d (1)-FIG. 3d (4)). Two ratsimmunized with synthetic a.a.123-134 human IFN_(r) peptide conjugatedwith OVA also developed antibodies to human IFN_(r), which couldcross-react with Aspergillus Noc2 (FIG. 3d (1)-FIG. 3d (4)). Moreover,it was found that all sera from rats immunized with Noc2 peptide as wellas those immunized with IFN_(r) peptide can block human recombinantIFN_(r) (FIG. 3e ). These data indicate that antibodies to AspergillusNoc2 could cross-react with human IFN_(r) in vivo and vice versa.

Next, the inventor checked if these Noc2 antibodies had a neutralizingability on the IFN_(r) signaling pathway. Using a p-STAT1 signalingassay by flow cytometry, it was observed that p-STAT1 signaling wasup-regulated in THP1 cells while pre-incubated with 50% sera fromnon-immunized rats and stimulated with recombinant IFN_(r). However,decreased up-regulation of p-STAT1 signaling in THP1 cells was observedwhile pre-incubated with 50% Noc2 or IFN_(r) immunized rats sera wasstimulated with recombinant IFN_(r) (FIG. 3f ). Similar results werealso be observed in an HLA-DR expression assay (FIG. 3g ). These dataindicate that antibodies to Aspergillus Noc2 not only bind to humanIFN_(r), but also inhibit IFN_(r) downstream signaling. Thus, ratsimmunized with Noc2 had a break in the immunological tolerance toIFN_(r) and anti-IFN_(r) antibodies were evoked through the process ofmolecular mimicry.

[Embodiment X] EE-IFN_(r) Application in Anti-IFN_(r) AutoAbs Therapy ExVivo

The inventor showed that EE-IFN_(r) could restore IFN_(r) activatedSTAT-1 signaling and promote IL-12/HLA-DR expression in controlPBMCs/THP1 cells with the presence of anti-IFN_(r) AutoAbs (FIGS. 2d-2f). Consequently, we investigated the possibility of using EE-IFN_(r) torestore the IFN_(r) function in patients with anti-IFN_(r) AutoAbs exvivo. Patients' PBMCs were pre-incubated with 10% autologous bloodplasma and stimulated with EE-IFN_(r) or BCG plus EE-IFN_(r) to measurep-STAT1 signaling or IL-12 production. It was observed that up-regulatedp-STAT1 signaling cannot be observed when IFN_(r) AutoAbs patients'PBMCs are pre-incubated with 10% autologous blood plasma and stimulatedwith recombinant IFN_(r) and WT IFN_(r) _(_)1-131. In contrast,EE-IFN_(r) could restore p-STAT1 signaling in four of six IFN_(r)AutoAbs patients' PBMCs pre-incubated with 10% autologous blood plasma(FIGS. 4a-4b ). Moreover, BCG plus EE-IFN_(r) could restore IL-12p40production in three of six IFN_(r) AutoAbs patients' PBMCs pre-incubatedwith 10% autologous blood plasma in contrast to BCG plus recombinantIFN_(r) or plus WT IFN_(r) _(_)1-131 (FIG. 4c ). The similar phenomenawas also be observed in an HLA-DR expression assay. It was found thatup-regulated HLA-DR expression levels were not observed when IFN_(r)AutoAbs patients' PBMCs was pre-incubated with 10% autologous bloodplasma and stimulated with recombinant IFN_(r) and WT IFN_(r) _(_)1-131.In contrast, EE-IFN_(r) could restore the HLA-DR expression levels infour of six IFN_(r) AutoAbs patients' PBMCs that were pre-incubated with10% autologous plasma (FIGS. 4d-4e ).

Next, the inventor examined EE-IFN_(r) bioactivity to restore IFN_(r)receptor activation in IFN_(r) AutoAbs patients' whole blood ex vivo.The p-STAT1 signal was up-regulated in controls' whole blood after beingstimulated with recombinant commercial IFN_(r), WT IFN_(r) _(_)1-131 orEE-IFN_(r). The up-regulated p-STAT1 signaling was not found whenIFN_(r) AutoAbs patients' whole blood was stimulated with recombinantIFN_(r) and WT IFN_(r) _(_)1-131. In contrast, EE-IFN_(r) could restorep-STAT1 signaling in four of six IFN_(r) AutoAbs patients' whole blood(FIGS. 4f-4g ). Moreover, BCG plus EE-IFN_(r) could restore IL-12p40production in three of six IFN_(r) AutoAbs patients' whole blood incontrast to BCG plus commercial IFN_(r) or plus WT IFN_(r) _(_)1-131R(FIG. 4h ). Taking these data together, EE-IFN_(r) could restorebioactivity not only in IFN_(r) AutoAbs patients' autologous plasma butalso in their whole blood ex vivo.

In conclusion, the inventor identified a major epitope in the C-terminalof IFN_(r) recognized by anti-IFN_(r) AutoAbs, and these AutoAbs couldcross-react with Aspergillus Noc2 protein. The inventor hypothesizesthat in the presence of specific HLA class II alleles, neutralizinganti-IFN_(r) AutoAbs, which recognized a limited epitope P₁₂₁₋₁₃₁ in theC-terminal of IFN_(r), were triggered by Noc2 protein through themechanism of molecular mimicry. Moreover, the inventor generated apotential therapeutic EE-IFN_(r), which could restore the IFN_(r)signaling pathway in the presence of patients' blood samples ex vivo.These data suggest that the anti-IFN_(r) AutoAbs to P₁₂₁₋₁₃₁ is the oneor the only crucial AutoAbs which causes this disease. These findingsprovide a new model for the pathogenesis of disseminated mycobacterialinfections caused by anti-IFN_(r) AutoAbs and a new therapeutic strategyfor this devastating disease.

Further Embodiments

1. A method for evaluating an efficacy of an isolated recombinant humaninterferon gamma (hIFN_(r)) for regulating a peripheral bloodmononuclear cell (PBMC), comprising the steps of:

-   -   providing the PBMC from a subject with anti-interferon gamma        autoantibodies;    -   mixing the isolated recombinant human interferon gamma with the        PBMC, wherein the isolated recombinant human interferon gamma        contains a homologous substitute; and    -   evaluating the efficacy of the isolated recombinant human        interferon gamma according to an expression level of a        phosphorylation of signal transducers and activators of        transcription 1 (p-STAT1) generated by the PBMC.        2. The method as claimed in Embodiment 1, wherein the isolated        recombinant human interferon gamma is produced by the following        steps:    -   determining an epitope of a subject suffering from a        disseminated mycobacterial infection, the epitope is chosen from        at least one residue in a human interferon gamma; and    -   applying a mutation procedure to the at least one residue to        activate a receptor of the human interferon gamma which is not        neutralized by an autoantibody of the human interferon gamma.        3. The method as claimed in Embodiment 1, wherein the        recombinant human interferon gamma is characterized by an        epitope of a subject suffering from a disseminated mycobacterial        infection; and    -   the homologous substitute substitutes for at least one residue        in the epitope.        4. The method as claimed in Embodiment 3, wherein the epitope is        located between residue 121 and 127 from a C-terminal of a human        interferon gamma.        5. The method as claimed in Embodiment 3, wherein the homologous        substitute includes a peptide “Leu-Pro-Glu-Ser-Ser-Leu-Arg” (SEQ        NO: 1).        6. The method as claimed in Embodiment 3, wherein the        recombinant human interferon gamma further includes one of a        pharmaceutically acceptable excipient and a pharmaceutically        acceptable carrier.        7. The method as claimed in Embodiment 3, wherein the        recombinant human interferon gamma up-regulates the expression        level of the p-STAT1 demonstrating a bioactivity of a        hIFN_(r)⋅⋅⋅        8. A method for evaluating an efficacy of an isolated        recombinant cytokine for regulating a peripheral blood        mononuclear cell (PBMC), comprising the steps of:    -   providing the PBMC from a subject with an anticytokine        autoantibody;    -   mixing the isolated recombinant cytokine with the PBMC, wherein        the isolated recombinant cytokine contains a homologous        substitute; and    -   evaluating the efficacy of the isolated recombinant cytokine        according to an expression level of an interleukin-12 (IL-12)        generated by the PBMC.        9. The method as claimed in Embodiment 8, wherein the isolated        recombinant human interferon gamma is produced by the following        steps:    -   determining an epitope of a subject suffering from a        disseminated mycobacterial infection, wherein the epitope lacks        at least one residue in the amino acid sequence; and    -   substituting the homologous substitute for the at least one        residue in the epitope.        10. The method as claimed in Embodiment 9, wherein the epitope        is located between residue 121 and 127 from a C-terminal of a        human interferon gamma.        11. The method as claimed in Embodiment 9, wherein the        homologous substitute includes a peptide        “Leu-Pro-Glu-Ser-Ser-Leu-Arg” (SEQ NO: 1).        12. A recombinant protein □, comprising:        a human interferon gamma having a sequence replaced by a peptide        of “Leu-Pro-Glu-Ser-Ser-Leu-Arg”, wherein the recombinant        protein is used to activate a receptor of an interferon gamma        (IFN_(r)) and is free from neutralization by an autoantibody of        the interferon gamma of a subject suffering from a disseminated        mycobacterial infection.        13. The recombinant protein as claimed in Embodiment 12, wherein        the subject is immunized with a human interferon gamma.        14. The recombinant protein as claimed in Embodiment 12, wherein        the subject is immunized with Aspergillus Noc2.        15. The recombinant protein as claimed in Embodiment 12, wherein        the sequence has a C-terminal and is located between residue 121        and 127 from the C-terminal.        16. The recombinant protein as claimed in Embodiment 15, wherein        the recombinant protein is characterized by an expression of a        phosphorylation of signal transducers and activators of        transcription 1 (p-STAT1).        17. The recombinant protein as claimed in Embodiment 15, wherein        the recombinant protein is characterized by an expression of an        interleukin-12 (IL-12).        18. The recombinant protein as claimed in Embodiment 15, wherein        the recombinant protein is characterized by an expression of        Human Leukocyte Antigen-antigen D Related (HLA-DR) expression.        19. The recombinant protein as claimed in Embodiment 15, wherein        the recombinant protein further includes one of a        pharmaceutically acceptable excipient and a pharmaceutically        acceptable carrier.        20. The recombinant protein as claimed in Embodiment 15, wherein        the C-terminal of the human interferon gamma is recognized by        the autoantibody of the interferon gamma.

While the invention has been described in terms of what is presentlyconsidered to be the most practical and preferred embodiments, it is tobe understood that the invention needs not be limited to the disclosedembodiments. On the contrary, it is intended to cover variousmodifications and similar arrangements included within the spirit andscope of the appended claims which are to be accorded with the broadestinterpretation so as to encompass all such modifications and similarstructures.

What is claimed is:
 1. A method for evaluating an efficacy of anisolated recombinant human interferon gamma (rhIFNγ) for regulating aperipheral blood mononuclear cell (PBMC), comprising the steps of:providing the PBMC from a subject with anti-interferon gammaautoantibodies; mixing the isolated recombinant human interferon gamma(rhIFNγ) with the PBMC, wherein the isolated recombinant humaninterferon gamma (rhIFNγ) is produced by replacing at least one residuewhich is located between residues 121 and 127 from a C-terminal of anepitope of a human interferon gamma (hIFNγ) SEQ ID NO: 39 via homologoussubstitution; and evaluating the efficacy of the isolated recombinanthuman interferon gamma (rhIFNγ) according to an expression level of aphosphorylation of signal transducers and activators of transcription 1(p-STAT1) generated by the PBMC.
 2. The method as claimed in claim 1,wherein homologous substitution consists of SEQ ID NO:
 1. 3. The methodas claimed in claim 2, wherein the isolated recombinant human interferongamma (rhIFNγ) further includes a pharmaceutically acceptable excipientor a pharmaceutically acceptable carrier.
 4. The method as claimed inclaim 2, wherein the isolated recombinant human interferon gamma(rhIFNγ) up-regulates the expression level of the p-STAT1.
 5. A methodfor evaluating an efficacy of an isolated recombinant human interferongamma (rhIFNγ) for regulating a peripheral blood mononuclear cell(PBMC), comprising the steps of: providing the PBMC from a subject withanti-interferon gamma autoantibodies; mixing the isolated recombinanthuman interferon gamma (rhIFNγ) with the PBMC, wherein the isolatedrecombinant human interferon gamma (rhIFNγ) is produced by replacingresidues which is located between residues 121 and 127 from a C-terminalof an epitope of a human interferon gamma (hIFNγ) of SEQ ID NO: 39 viahomologous substitution of SEQ ID NO: 1; and evaluating the efficacy ofthe isolated recombinant human interferon gamma (rhIFNγ) according to anexpression level of a phosphorylation of signal transducers andactivators of transcription 1 (p-STAT1) generated by the PBMC.